Abstract: Over the last five years, the characterization of the pluripotent state and its differentiated progeny has made remarkable progress. Previous studies have identified most of the building blocks participating in the establishment, maintenance and differentiation of the pluripotent state. However, most studies so far relied on correlation based evidence and inference, and did not yet obtain a functionally validated picture of its regulatory foundation. Here, we propose to overcome parts of this problem by addressing the following questions: Aim 1: What are the functionally relevant cis-regulatory modules (CRMs) and gene regulatory networks (GRNs) for pluripotency and differentiation initiation? Previous work has mapped the epigenetic state, transcription factor binding and RNA expression in pluripotent, reprogramming and differentiating cells, yielding a list of putative gene regulatory elements (GREs). Here, we will define the subset of functionally relevant GREs and CRMs for these cell types. To that end, we will employ massively parallel reporter assays (MPRA) to assess the functionality of thousands of GREs in parallel, identify their upstream transcriptional regulators through targeted motif mutagenesis and define their phenotypic relevance through epigenome editing mediated loss of function experiments for hundreds of GREs. Aim 2: What functional role does DNA methylation play in the regulation of CRMs in pluripotency and differentiation? While it is well established that changes in the DNA methylation patterns can demarcate GREs, it is less clear whether these changes are functionally relevant for the proper operation of the corresponding CRMs. To investigate the contextual relevance of DNA methylation changes, we will employ a modified version of our MPRA that allows for the identification of those GREs that operate in a methylation sensitive fashion. These experiments will yield insights into context specific role of DNA methylation in the control of cell type specific GRNs. Aim 3: What is the cis-regulatory code controlling long non-coding RNA (lncRNA) activity and localization in pluripotency and differentiation? Recent findings from the Project II of this proposal identified a set of lncRNAs critical for pluripotency and differentiation. However, their regulation on the DNA and RNA level remains elusive. In order to address this question, we will employ two distinct novel MPRA designs, one for DNA elements and one adapted to RNA elements. These experiments will define the cis-regulatory landscape of lncRNA regulation on the DNA and RNA level in pluripotency and differentiation.